Publication Date

2015

Document Type

Honors Thesis

Department

Biochemistry

Keywords

Filariasis, Filariasis-Prevention, Filariasis-Treatment., Tropical medicine., Genetic regulation, Transcription factors, Affinity chromatography, Lymphatic filariasis, Neglected tropical disease, Gene regulation

Abstract

Lymphatic filariasis (LF) is a mosquito-borne Neglected Tropical Disease (NTD) caused by Wuchereria bancrofti, Brugia malayi and Brugia timori, three species of filarial parasites. Endemic in 73 tropical and sub-tropical countries, LF puts 1.4 billion people at risk of infection. 120 million are infected with the disease, of which 40 million are severely disabled. LF manifests in asymptomatic, acute and chronic forms and patients commonly develop elephantiasis and lymphedema. Social and psychological problems are associated with this disease, due to its disfiguring nature. Furthermore, since it is disabling, LF reduces the workforce capable of performing labor-intensive jobs in developing countries. Thus, it has a severely negative economic impact, growing the cycle of poverty. Current treatments delivered by mass drug administration, through an initiative of the World Health Organization, are effective in eliminating the microfilarial stage of the disease-causing parasites, but are ineffective at eliminating adult worms. Moreover, no vaccine capable of producing sterilizing immunity exists. Thus, it is imperative to identify promising vaccine and drug candidates that can target the parasites at their infective stage, in order to eliminate disease. In B. malayi, the thioredoxin peroxidase-2 (tpx-2) gene is upregulated when the parasite switches host from mosquito to human. TPX-2 produced by the gene detoxifies harmful oxygen radicals and thiols secreted by the human immune system onto the parasite, thus protecting it from being destroyed upon entry into the new host. The mechanism used by the parasite to upregulate this gene during its infective stage is unknown, which demands a study of its promoter region. Eukaryotic promoters are complex, functioning through the binding of several DNA-binding proteins known as transcription factors. The purpose of this project is to develop an in vitro proof-of-principle technique that uses a six-step affinity chromatography method to identify unique tpx-2 promoterbinding proteins. Briefly, the technique first immobilizes the promoter sequence as bait on a magnetic bead matrix. This bait DNA is incubated with nuclear extract derived from B. malayi, following which promoter-binding proteins are eluted. The eluted proteins are visualized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and their identity elucidated using mass spectrometry. Results show that this method has promise for the identification of promoter-binding proteins. In future studies, it holds the potential to be further optimized and adapted as a robust method for transcription factor identification in filarial parasites. This project, thus, hopes to expand current knowledge about filarial transcription and provides a targeted approach to the identification of effective vaccine and drug targets in the battle against LF.

Language

English

Comments

97 pages : color illustrations, color maps. Honors project-Smith College, 2015. Includes bibliographical references (pages 91-97)

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